An invertebrate model for CNS drug discovery: Transcriptomic and functional analysis of a mammalian P-glycoprotein ortholog

•A simple and high-throughput locust-based in situ screening model was developed.•Gene and kinetic analyses revealed an efflux activity across locust brain barrier.•The locust model can predict drug brain disposition and P-glycoprotein substrates.

BackgroundABC efflux transporters at the blood brain barrier (BBB), namely the P-glycoprotein (P-gp), restrain the development of central nervous system (CNS) drugs. Consequently, early screening of CNS drug candidates is pivotal to identify those affected by efflux activity. Therefore, simple, high-throughput and predictive screening models are required. The grasshopper (locust) has been developed as an invertebrate in situ model for BBB permeability assessment, as it has shown similarities to vertebrate models.MethodsTranscriptome profiling of ABC efflux transporters in the locust brain was performed. Subsequently, identified transcripts were matched with their counterparts in human, rat, mouse and Drosophila melanogaster, based on amino acid sequence similarity, and phylogenetic trees were constructed to reveal the most likely evolutionary history of the proteins. Further, functional characterization of a P-gp ortholog was achieved through transport studies, using a selective P-gp substrate and locust brain in situ, followed by kinetic analyses.ResultsA protein with high sequence similarity to the ABCB1 gene of vertebrates was found in the locust brain, which encodes P-gp in human and is considered the most vital efflux pump. Functionally, this model showed transport kinetic behaviors comparable to those obtained from in vitro models. Particularly, substrate affinity of the putative P-gp was observed as in P-gp expressing cells lines, used for predicting drug penetration across biological barriers.ConclusionFindings suggest a conserved mechanism of brain efflux activity between insects and vertebrates, confirming that this model holds promise for inexpensive and high-throughput screening relative to in vivo models, for CNS drug discovery.

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